Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing 3-hydroxyisobutyrate or methacrylic acid related thereto

Patent metadata

Field	Value
Publication number	US-9346902-B2
Application number	US-201314071339-A
Country	US
Kind code	B2
Filing date	Nov 4, 2013
Priority date	Nov 5, 2012
Publication date	May 24, 2016
Grant date	May 24, 2016

Abstract

Provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway that can enhance the availability of reducing equivalents in the presence of methanol. Such reducing equivalents can be used to increase the product yield of organic compounds produced by the microbial organism, such as 3-hydroxyisobutyrate or MAA. Also provided herein are methods for using such an organism to produce 3-hydroxyisobutyrate or MAA.

First claim

What is claimed is:

1. A non-naturally occurring E. coli comprising
(A) a methanol metabolic pathway having a methanol dehydrogenase and at least one exogenous nucleic acid encoding a methanol dehydrogenase expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol, and
(B) a 3-hydroxyisobutyrate pathway comprising
(1) (i) a succinyl-CoA transferase, ligase, or synthetase; (ii) a methylmalonyl-CoA mutase; (iii) a methylmalonyl-CoA epimerase; (iv) a methylmalonyl-CoA reductase (aldehyde forming); and (v) a methylmalonate semialdehyde reductase;
(2) (i) a succinyl-CoA transferase, ligase, or synthetase; (ii) a methylmalonyl-CoA mutase; (iii) a methylmalonyl-CoA reductase (aldehyde forming); and (iv) a methylmalonate semialdehyde reductase; or
(3) (i) a succinyl-CoA transferase, ligase, or synthetase; (ii) a methylmalonyl-CoA mutase; and (iii) a methylmalonyl-CoA reductase (alcohol forming).

2. The E. coli of claim 1 , wherein the methanol metabolic pathway comprises:
(i) a methanol dehydrogenase, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate deformylase;
(ii) a methanol dehydrogenase, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate synthetase;
(iii) a methanol dehydrogenase and a formaldehyde dehydrogenase;
(iv) a methanol dehydrogenase, a S-(hydroxymethyl)glutathione synthase, a glutathione-dependent formaldehyde dehydrogenase and a S-formylglutathione hydrolase;
(v) a methanol dehydrogenase, a glutathione-dependent formaldehyde dehydrogenase and a S-formylglutathione hydrolase;
(vi) a methanol dehydrogenase, a formaldehyde activating enzyme, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate deformylase; or
(vii) a methanol dehydrogenase, a formaldehyde activati ng enzyme, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate synthetase.

3. The E. coli of claim 2 , wherein the methanol metabolic pathway further comprises (i) a formate dehydrogenase; (ii) a formate hydrogen lyase; or (iii) a formate hydrogen lyase and an hydrogenase.

4. The E. coli of claim 1 , further comprising at least two, three, four, five, six or seven exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme.

5. The E. coli of claim 4 , wherein at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme is a heterologous nucleic acid.

6. The E. coli of claim 1 , further comprising at least one, two, three, four, five, six or seven exogenous nucleic acids, each encoding a 3-hydroxyisobutyrate pathway enzyme expressed in a sufficient amount to produce 3-hydroxyisobutyrate.

7. The E. coli of claim 4 , wherein said at least one exogenous nucleic acid encoding a 3-hydroxyisobutyrate pathway enzyme is a heterologous nucleic acid.

8. The E. coli of claim 1 comprising one or more gene disruptions, wherein said one or more gene disruptions occur in one or more endogenous genes encoding protein(s) or enzyme(s) involved in native production of ethanol, glycerol, acetate, lactate, formate, CO 2 , and/or amino acids, by said E. coli , and wherein said one or more gene disruptions confers increased production of 3-hydroxyisobutyrate in said E. coli.

9. The E. coli of claim 1 , wherein one or more endogenous enzymes involved in: native production of ethanol, glycerol, acetate, lactate, formate, CO 2 and/or amino acids by said E. coli , has attenuated enzyme activity or expression levels.

10. The E. coli of claim 1 further comprising a formaldehyde assimilation pathways and at least one exogenous nucleic acid encoding a formaldehyde assimilation pathway enzyme expressed in a sufficient amount to produce an intermediate of glycolysis and/or a metabolic pathway that can be used in the formation of biomass, and wherein
(a) said formaldehyde assimilation pathway comprises a hexulose-6-phosphate synthase and a 6-phospho-3-hexuloisomerase; or
(b) said formaldehyde assimilation pathway comprises a dihydroxyacetone synthase and a dihydroxyacetone kinase.

11. The E. coli of claim 10 wherein the intermediate is (i) a hexulose-6-phosphate, a fructose-6-phosphate, or a combination thereof; or (ii) a dihydroxyacetone, a dihydroxyacetone phosphate, or a combination thereof.

12. The E. coli of claim 1 , wherein said E. coli is in a substantially anaerobic culture medium.

13. A method for producing 3-hydroxyisobutyrate, comprising culturing the E. coli of claim 1 under conditions and for a sufficient period of time to produce 3-hydroxyisobutyrate.

14. The method of claim 13 further comprising separating the 3-hydroxyisobutyrate from other components in the culture.

15. The method of claim 14 wherein the separation comprises extraction, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, or ultrafiltration.

16. The method of claim 13 , wherein the E. coli is cultured in a medium comprising biomass, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, starch, glycerol, methanol, carbon dioxide, formate, methane, or any combination thereof as a carbon source.

17. The method of claim 13 , wherein the E. coli is cultured in a substantially anaerobic culture medium.

18. The method of claim 13 , wherein the E. coli is cultured in a culture medium comprising glucose.

Assignees

• Genomatica Inc

Inventors

• Burgard Anthony P
• Osterhout Robin E
• Van Dien Stephen J
• Tracewell Cara Ann
• Pharkya Priti
• Andrae Stefan

Classifications

• C08G63/664

  derived from hydroxy carboxylic acids · CPC title

• C12P7/24

  containing a carbonyl group · CPC title

• C12P7/42

  Hydroxy-carboxylic acids · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N15/52 Primary

  Genes encoding for enzymes or proenzymes · CPC title